epigenetics and its implications for ecotoxicology · epigenetics and its implications for...

Epigenetics and its implications for ecotoxicology

Michiel B. Vandegehuchte • Colin R. Janssen

Accepted: 5 March 2011 / Published online: 22 March 2011

� Springer Science+Business Media, LLC 2011

Epigenetics is the study of mitotically or meiotically her-

itable changes in gene function that occur without a change

in the DNA sequence. Interestingly, epigenetic changes can

be triggered by environmental factors. Environmental

exposure to e.g. metals, persistent organic pollutants or

endocrine disrupting chemicals has been shown to modu-

late epigenetic marks, not only in mammalian cells or

rodents, but also in environmentally relevant species such

as fish or water fleas. The associated changes in gene

expression often lead to modifications in the affected

organism’s phenotype. Epigenetic changes can in some

cases be transferred to subsequent generations, even when

these generations are no longer exposed to the external

factor which induced the epigenetic change, as observed in

a study with fungicide exposed rats. The possibility of this

phenomenon in other species was demonstrated in water

fleas exposed to the epigenetic drug 5-azacytidine. This

way, populations can experience the effects of their

ancestors’ exposure to chemicals, which has implications

for environmental risk assessment. More basic research is

needed to assess the potential phenotypic and population-

level effects of epigenetic modifications in different species

and to evaluate the persistence of chemical exposure-

induced epigenetic effects in multiple subsequent

generations.

Keywords DNA methylation � Transgenerational

effects � Invertebrates � Environmental toxicology

Introduction

Epigenetics is the study of heritable changes in gene func-

tion that occur without a change in the DNA sequence which

forms the genetic code, hence the prefix ‘epi’, from the

Greek word meaning ‘above, beyond’. Several reviews have

dealt with the subject, each with a specific line of approach:

e.g. molecular targets of epigenetic mechanisms, transgen-

erational inheritance of epigenetic modifications, conse-

quences of epigenetic changes for safety assessment, animal

models to study epigenetics, epigenetic impacts of adverse

environmental effects in mammals (Rosenfeld 2010;

Choudhuri et al. 2010; Jablonka and Raz 2009; LeBaron

et al. 2010; Bollati and Baccarelli 2010). Toxicant exposure

can perturb the epigenetic status of cells and organisms, as

known from several studies on cell lines or rodent models.

Since a perturbed epigenetic status can entail changes in

gene expression levels, which may have effects on organ-

isms and populations, the study of epigenetics is also highly

relevant for ecotoxicologists. In ecotoxicology however,

epigenetic research is still very limited. Therefore, the aims

of the current review are (1) to give a brief background of

epigenetics and epigenetic mechanisms and (2) to summa-

rize and discuss the current literature on environmental

impacts on epigenetics, with a focus on toxicant exposure,

highlighting the present knowledge and research needs

related to epigenetics in ecotoxicology.

Epigenetics

Definition

Various authors have provided a definition for the con-

stantly evolving science of epigenetics. The term was

M. B. Vandegehuchte (&) � C. R. Janssen

Laboratory of Environmental Toxicology and Aquatic Ecology,

Ghent University (UGent), Jozef Plateaustraat 22,

9000 Ghent, Belgium

e-mail: [email protected]

123

Ecotoxicology (2011) 20:607–624

DOI 10.1007/s10646-011-0634-0

introduced by Conrad Waddington (1939) to describe ‘the

causal interactions between genes and their products,

which bring the phenotype into being’. This was mainly

used in the context of unfolding the genetic program for

development. In the last decade of the twentieth century,

scientists realized that epigenetics had broader implications

and two new definitions of epigenetics were proposed

(Holliday 1994): ‘The study of the changes in gene

expression which occur in organisms with differentiated

cells, and the mitotic inheritance of given patterns of gene

expression’ and ‘Nuclear inheritance which is not based on

changes in DNA sequence’. The first definition is rather

broad, stating nothing about molecular mechanisms, but

stressing the importance of mitotic inheritance, which is

the inheritance of an epigenetic status between cells after

normal mitotic cell division. In the second definition, the

evolution in the genetic field since Waddington’s paper is

obvious: it points to mechanisms not related to changes in

the sequence of the four known DNA bases. Based on

Holliday’s two definitions, a new definition was put for-

ward by Riggs et al. (1996). Variants of this working

definition are still used in current textbooks and by many

scientists in the field (Allis et al. 2007; Berger et al. 2009).

It is also this definition that will be used in this review:

‘Epigenetics is the study of mitotically and/or meiotically

heritable changes in gene function that cannot be explained

by changes in DNA sequence’.

Mechanisms

Several molecular mechanisms are known to be involved in

epigenetics. Next to the three important and well-studied

systems that are described below, new molecular aspects of

epigenetic control have very recently been discovered and

are subject of intensive research nowadays, e.g. cytosine

hydroxymethylation and nucleosome-space occupancy

(Tahiliani et al. 2009; Cui et al. 2010).

DNA methylation

DNA methylation in eukaryotes occurs through the transfer

of a methyl group from S-adenosyl-L-methionine (SAM) to

the 5-position of cytosine, to form 5-methylcytosine

(Fig. 1a). In mammals this mainly occurs in a CpG

(deoxycytidine linked with a phosphate group to

deoxyguanosine) dinucleotide context, while in plants

methylation is also found at CZG or CZZ sites (with

Z = A, C or T for deoxyadenosine, deoxycytidine or

thymidine, respectively, with all neighboring nucleosides

linked by a phosphate group) (Zhang 2008). Methylated

cytosines can be deaminated and transformed into thymi-

dine. As a result of this C to T transition mutation, which is

difficult to repair, methylated CpGs are expected to

decrease in abundance with evolutionary time, resulting in

CpG deficiency. The ratio of observed to expected CpGs

can be used to predict methylated and unmethylated

genomic regions (Wang and Leung 2008). The enzymes

responsible for DNA methylation are DNA methyltrans-

ferases or DNMTs. In humans, three groups of DNMTs are

described. DNMT1 is the maintenance methyltransferase.

DNMT3 enzymes establish methylation patterns in a

totally unmethylated CpG context (de novo methylation).

DNMT2, while possessing a weak DNA methyltransferase

activity, is actually also involved in tRNA methylation

(Jurkowski et al. 2008; Jeltsch et al. 2006). Adenine

methylation is known to occur in bacteria (Collier 2009),

however, discussion of this mechanism is beyond of the

scope of this review.

A

B

C

Fig. 1 Schematic diagram of three main epigenetic mechanisms.

a DNA methylation by the transfer of a methyl group (–CH3) to the

5-position of cytosine, to form 5-methylcytosine. SAM: S-adenosyl-

methionine, SAH: S-adenosylhomocysteine, DNMT: DNA-methyl-

transferase. Chemicals may interact by e.g. using SAM as a methyl

donor in their metabolisation or inhibiting the action of DNMTs.

b Histone deacetylation and histone methylation represent histone tail

modifications. HDAC: Histone deacetylase, HMT: histone methyl-

transferase. Chemicals may interact with the action of both HDACs

and HMTs. c Non-coding RNA molecules such as microRNA

(miRNA) and small interfering RNA (siRNA) are formed from

double-stranded RNA (dsRNA) or hairpin RNA molecules. The

RNA-induced silencing complex (RISC) is targeted to complemen-

tary messenger RNA (mRNA) or DNA and interferes with translation

or transcription

608 M. B. Vandegehuchte, C. R. Janssen

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Levels and patterns of DNA methylation can differ

substantially between different species. The nematode

worm Caenorhabditis elegans does not possess a conven-

tional DNA methyltransferase and completely lacks

detectable DNA methylation (Bird 2002). The fruit fly

Drosophila melanogaster was also long thought to have an

unmethylated genome. However, now it is known that it

possesses a DNA-methyltransferase-like gene. A low level

of methylation was observed in its genome: 0.1–0.4% of

the cytosines were methylated, with the highest levels

noted in early-stage embryos. An additional peculiarity is

that the methylation in D. melanogaster is mostly found in

CpT and CpA dinucleotides, as opposed to the CpG

dinucleotide context of vertebrate DNA methylation

(Tweedie et al. 1999; Lyko 2001). In the genome of the

honey bee Apis mellifera and the wasp Nasonia vitripennis,

homologs to all three types of DNMTs were found and

CpG methylation has been observed in several genes.

However, the overall DNA methylation level has not been

quantified yet (Wang et al. 2006; Schaefer and Lyko 2007;

The Nasonia Genome Working Group 2010). CpG meth-

ylation has also been detected in DNA of the water flea

Daphnia magna (Vandegehuchte et al. 2009a). In this

species, 0.22–0.35% of the cytosines are methylated under

standard conditions (Vandegehuchte et al. 2009b). Other

invertebrate genomes display moderate CpG methylation

levels, often in a pattern known as mosaic methylation:

methylated cytosines concentrated in large domains, sep-

arated by domains with unmethylated DNA. In the sea

urchin Strongylocentrotus purpuratus and the sea squirt

Ciona intestinalis, roughly half of the genome consists of

DNA domains in which most of the CpGs are methylated,

while the remaining DNA is methylation-free (Tweedie

et al. 1997; Suzuki et al. 2007). In vertebrates, the meth-

ylation level is high: approximately 5% of the cytosines are

methylated in DNA of mammals and birds and roughly

10% in that of fish and amphibians (Field et al. 2004). This

methylation is spread evenly throughout almost the entire

genome with the notable exception of short CpG-rich

regions named CpG islands, found at the 50 position of a

large number of genes and which are generally unmethy-

lated (Suzuki and Bird 2008). These different methylation

distributions suggest that DNA methylation may have

different functions in different organisms.

In vertebrates, DNA methylation is generally associated

with silencing of genes or maintaining a silenced state. In

the past decade a consensus view had appeared that DNA

methylation is a mediator of transcriptional silencing. This

was mainly based on studies in CpG islands of gene pro-

moters in mammalian DNA. However, recent data on

vertebrate DNA, as well as some less known studies of

invertebrate DNA methylation, suggest a broader role of

this epigenetic mark. Gene body methylation (as opposed

to promoter methylation) of the Ddah2 gene was shown to

be a biomarker for embryonic stem cell differentiation to

neural stem cells (Backdahl et al. 2009). Remarkable in this

study is that DNA methylation was positively correlated

with gene expression. In different clones of the aphid

Myzus persicae, gene body methylation was shown to play

a role in the transcription of E4 genes, coding for insecti-

cide-detoxifying esterase proteins. As in the study of

Backdahl et al., methylated genes were expressed, while

loss of the methylation was correlated with a loss of tran-

scription (Field 2000). Bongiorni et al. (1999) demon-

strated that unmethylated chromosomes of the mealybug

Planococcus citri were inactivated by heterochromatini-

zation, while methylated chromosomes remained active. In

the genome of the honey bee Apis mellifera, CpG meth-

ylation is suggested to be used as an epigenetic mechanism

to maintain transcription of ‘housekeeping genes’, neces-

sary for conserved core biological processes in virtually

every type of cell (Foret et al. 2009). A newer hypothesis

for the functional role of DNA methylation, supported by

the above described data, is the prevention of transcription

initiation (Zilberman 2008; Suzuki and Bird 2008). This

preserves the idea that DNA methylation is a transcrip-

tional repressor, but allows intragenic methylation in an

active transcription unit: i.e. it does not preclude tran-

scription as such, but rather its (spurious) initiation.

Moreover, this concept is in line with the cyclical DNA

methylation/demethylation which has been described in

transcriptional cycling of a gene in a human cell line

(Metivier et al. 2008).

Histone tail modifications or the histone code

The genome is organized in chromatin, consisting of DNA

and associated proteins. The building blocks of chromatin

are nucleosomes, which consist of approximately 180–190

base pairs, of which the majority is coiled twice around an

octamer of histone (H) proteins (four times two molecules

of H2A, H2B, H3 and H4). The linker histone protein H1 at

the outside of the nucleosome serves to further compact the

chromatin (Pruss et al. 1995). The structure of these his-

tones is not static. Their mutual affinity as well as their

affinity for DNA and for other chromatin associated pro-

teins is determined by post-translational modifications of

their protruding amino-terminal tails. These modifications

may include acetylation, ubiquitination, phosphorylation or

methylation (Fig. 1b). Together, they form a histone code,

which affects gene transcription and chromatin structure

(Lennartsson and Ekwall 2009). These modifications are

performed by specialized enzymes such as histone acetyl

transferases (HAT), histone methyl transferases (HMT) and

histone deacetylases (HDAC). Active histone marks, e.g.

acetylation of H3 on lysine 9 (H3K9) or dimethylation of

Epigenetics and its implications for ecotoxicology 609

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H3K4, lead to chromatin decondensation and thus result

in the formation of euchromatin, creating conditions for

gene transcription. Conversely, condensed heterochromatin

lacks this histone acetylation and is enriched in trimethy-

lated lysine 9 and lysine 27 on H3 (Bartova et al. 2008).

Two important types of proteins can be found in associa-

tion with specific histone modifications: the trithorax group

proteins and the polycomb group proteins, which are

associated with transcriptionally active euchromatin and

transcriptionally silent heterochromatin, respectively

(Schuettengruber et al. 2007).

It is known that there is interaction between histone

modifications and DNA methylation, although the precise

mechanisms have not been completely discovered. In a

study with knock-out mice lacking a DNA methyltrans-

ferase, it was shown that histone methylation in certain

genomic regions was markedly decreased (Henckel et al.

2009). This demonstrated that DNA methylation is

involved in the establishment and/or maintenance of his-

tone methylation in those regions. Methylated DNA can

also cause histone deacetylation, through binding with

proteins such as MeCP2, which interact with histone

deacetylases (Nan et al. 1998). Fuks (2005) on the other

hand, suggested that DNA methylation is a secondary

epigenetic modification by DNA methyltransferase

enzymes which are attracted to locations where histone H3

is methylated at lysine 9. It has also been shown that the

methylation of H3K27 was necessary for the recruitment of

DNA methyltransferases to methylate DNA in the pro-

moters of certain genes (Vire et al. 2006). Future research

will further elucidate the interactions and crosstalk between

the histone code and the DNA methylation machinery.

Non-coding RNA (ncRNA)

Gene silencing through small (approximately 20–30

nucleotides) non-coding RNA molecules most probably

originated as a protection system against invasive sequen-

ces such as transposons and viruses (Zilberman and

Henikoff 2005). ‘Non-coding’ refers to the fact that these

RNA strands do not function as messenger RNA, transfer

RNA or ribosomal RNA. The process of gene expression

regulation by small ncRNA molecules is also known as

RNA interference or RNAi (Yu 2008). The two main cat-

egories of these regulatory small RNAs are microRNA

(miRNA) and short interfering RNA (siRNA). These small

RNAs are excised from larger, double-stranded or hairpin-

shaped precursor molecules by a Dicer enzyme. They

associate with effector proteins (Argonaute proteins) to

form an RNA-induced silencing complex (RISC), which is

targeted to complementary nucleic acid sequences

(Fig. 1c). Silencing of genes can happen through tran-

scriptional or translational inhibition, heterochromatin

formation or DNA/RNA degradation (Carthew and Sont-

heimer 2009).

The RNA silencing system is also known to interact

with other epigenetic mechanisms. In association with

RNA polymerase, RISC can attract or activate DNA

methyltransferases (DNMTs), resulting in methylation of

DNA. This is a known mechanism of siRISC silencing

in plants, but also occurs in mammals (Lippman and

Martienssen 2004). RNAi is also suggested to play a role in

the protection of genomes against long-term epigenetic

defects, targeting remethylation of specific sequences

(Teixeira et al. 2009). In the yeast Saccharomyces pombe it

was discovered that the siRISC, also termed RNA-induced

transcriptional silencing complex (RITS), guides a histone

methyltransferase to methylate H3K9, ultimately resulting

in heterochromatin formation. This process also takes place

in plants and mammals (Wassenegger 2005).

Environmental factors and epigenetics

Introduction

Several types of environmental factors are known to affect

the epigenetic status of organisms and changes in all

known epigenetic mechanisms can be triggered by envi-

ronmental cues. Recently, alterations in DNA methylation

and gene expression due to space flight, a complex envi-

ronmental condition involving e.g. cosmic radiation,

microgravity and space magnetic fields, were demonstrated

in the rice plant Oryza sativa (Ou et al. 2009). Maternal

care was shown to affect DNA methylation, histone acet-

ylation and behavior of the offspring in a study of the

licking-grooming behavior of maternal rats toward their

pups (Weaver et al. 2004).

The notion that diet can influence epigenetics originated

in studies with children born during the Dutch Hunger

Winter in the Second World War. Adults who were

exposed to the famine as children in utero exhibited less

DNA methylation at a locus in the insulin like growth

factor gene and a larger incidence of coronary heart disease

and obesity, compared to their non-exposed siblings

(Heijmans et al. 2008). Laboratory studies with rats and

primates demonstrated that unbalanced prenatal nutrition

can induce histone acetylation changes or persistent, gene-

specific epigenetic changes which affect gene transcription

(Aagaard-Tillery et al. 2008; Lillycrop et al. 2005).

Also in non-mammalian species, nutrition can dramati-

cally affect the phenotype by inducing epigenetic changes.

In a honey bee (Apis mellifera) colony, fertile queens and

sterile workers develop from genetically identical larvae.

Larvae destined to become queens are fed with royal jelly,

while worker larvae get a less complex diet. Kucharski


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et al. (2008) demonstrated, in workers versus queens, dif-

ferent degrees of DNA methylation at several loci. More-

over, by silencing the DNA methyltransferase Dnmt3 in

newly-hatched larvae, they induced the development to

queens in the majority of these larvae. Thus, these results

suggest that DNA methylation stores epigenetic informa-

tion, which can be altered by nutrition and which has a

profound effect on the development and the adult pheno-

type of the honey bee. The impact of nutrition on epige-

netics can be relevant to ecotoxicological testing, where

organisms exposed to chemicals often need to be fed dur-

ing the exposure. However, to date this is an unexplored

area of research.

Transgenerational epigenetic effects

A very interesting aspect of epigenetic changes is the fact

that they can, in some cases, be inherited by subsequent

generations which are not exposed to the environmental

factor that caused the change. Several recent reviews have

described this phenomenon, each from a slightly different

point of view and with own interpretations of ‘transgen-

erational transfer’ or ‘inheritance’ (Jablonka and Raz 2009;

Ho and Burggren 2010). In this section, some studies on

the transgenerational transfer of epigenetic marks and their

alterations due to environmental factors will be discussed.

DNA methylation profiles are transferred through

somatic cell divisions by the action of maintenance DNA

methyltransferases. In sexually reproducing organisms,

epigenetic variations in DNA methylation should survive

the complex process of meiosis in order to be transmitted to

the next generation. In multicellular organisms, they should

also be maintained after gametogenesis and embryogene-

sis, resisting the significant restructuring of cells and

chromatin which takes place at these developmental stages.

In mammalian development, DNA is progressively deme-

thylated during the preimplantation period, after which

remethylation takes place (Bocock and Aagaard-Tillery

2009). Developmental DNA methylation changes have also

been demonstrated in the fruit fly Drosophila melanogaster

and the zebrafish Danio rerio (MacKay et al. 2007). There

is evidence that changed DNA methylation patterns can be

transmitted over generations in spite of these develop-

mental alterations, but at present it is not clear how this

occurs mechanistically (Bonduriansky and Day 2009).

Some authors argue that gametic inheritance of an epige-

netic change induced by exposing a gestating female to

environmental stress can only be proven if this change is

still present in the F3 generation. Indeed, the primordial

germ cells creating the F2 generation are already present in

the F1 embryo inside the pregnant F0 female (Youngson

and Whitelaw 2008). However, if an epigenetically

induced phenotype in the non-exposed F2 generation is

observed after exposure of F0 pregnant females, epigenetic

patterns must have been transferred between cells in the F2

generation. In that respect, this can also be called a trans-

generational epigenetic effect.

The occurrence of transgenerational epigenetic effects

has inspired several authors to study the impact of epige-

netics on evolution. In a somewhat provocative commen-

tary, Martienssen (2008) used the term ‘adaptive

Lamarckian evolution’, referring to epigenetic mechanisms

that may enable the environment to direct evolution at a

short time scale. In a breakthrough study with Arabidopsis,

Johannes et al. (2009) created a panel of epigenetic

Recombinant Inbred Lines, with little DNA sequence dif-

ferences, but contrasting DNA methylation profiles. Stable

inheritance of multiple parental DNA methylation variants

was demonstrated over at least eight generations, associ-

ated with heritability of flowering time and plant height.

This research demonstrated the relevance of incorporating

epigenetic information in population genetics studies. It

should be clear that epigenetics does not deny or reject

classic Darwinian evolution or genetics, but rather adds an

extra dimension to it. This is nicely illustrated in a study of

wild populations of the Spanish violet Viola cazorlensis,

showing epigenetic differentiation of different populations

at the DNA methylation level, which was correlated with

adaptive genetic divergence demonstrated by a population-

genomic analysis (Herrera and Bazaga 2010). In the con-

text of ecotoxicology, a knowledge gap that requires ded-

icated study concerns the potential of chemicals-induced

epigenetic modifications to be stably transmitted over

several generations. This could lead to epigenetic micro-

evolution, with e.g. local adaptation to stress, as suggested

by Morgan et al. (2007). In population studies of micro-

evolution due to chemical stress exposure, it would be

useful to screen for epigenetic modifications, next to

studying genetic adaptation.

A special model organism for studying (transgenera-

tional) environmental effects on DNA methylation is the

viable yellow agouti (Avy) mouse. In the Avy locus of this

model system genome, a transposon is associated with the

promoter region of the agouti gene, which codes for coat

color. When the agouti gene is fully expressed, these inbred

mice have yellow fur and suffer from obesity, diabetes and

increased tumor susceptibility. However, expression of this

gene depends on the degree of methylation in the Avy

locus. When this locus is hypermethylated, mice will be

brown or ‘pseudoagouti’. A hypomethylated Avy locus will

result in a yellow coat color, while partial methylation is

reflected in slightly or more densely ‘mottled’ fur color,

depending on the degree of methylation. As such, this

model allows for direct phenotypic assessment of the epi-

genetic DNA methylation state at the Avy locus (which is

called a metastable epiallele). Interestingly, mice that were


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fed a diet enriched with the methyl donors folic acid,

vitamin B12, choline and betaine exhibited a shift in phe-

notype in their offspring due to increased DNA methyla-

tion at the Avy locus (Waterland and Jirtle 2003). Similar

results were obtained in another mouse model, where

methylation of the AxinFu metastable epiallele determines

the shape of the tail: from straight to very kinky. Feeding

female mice a methyl donor-rich diet resulted in a clear

shift towards the straight tail phenotype in the offspring

(Waterland et al. 2006).

The naturally occurring DNA-methylation-dependent

variable phenotypes of Avy and AxinFu mice can be

transmitted to the subsequent generation. Morgan et al.

(1999) demonstrated that the phenotype of an Avy dam

influenced the phenotype distribution of the offspring. For

the AxinFu mouse, Rakyan et al. (2003) also demonstrated

that the phenotype of the parent influenced the phenotypic

distribution of the offspring. In this case, both the maternal

and the paternal phenotype had an effect. The fact that

mature sperm had the same methylation state at AxinFu as

the somatic tissue was consistent with the transgenerational

inheritance of epigenetic marks. The above described

phenotype shift in the F1 offspring of F0 females which

received nutritional methyl donor supplementation during

pregnancy was shown to be transferred to the F2 genera-

tion, although methyl donor supplemented food was only

given to F0 organisms (Cropley et al. 2006). The results of

this study were later questioned by Waterland et al. (2007).

These authors claim that diet-induced hypermethylation at

the Avy epiallele is not inherited transgenerationally, based

upon the absence of a cumulative effect of dietary methyl

donor supplementation across generations. However, diet

type and timing, as well as the initial epigenetic state of the

Avy allele differed between those studies. The results of

both studies have recently been interpreted as follows: a

methyl-donor supplemented diet can affect a female’s

propensity to transmit the Avy epiallele, although this does

not appear to engender novel epialleles at the Avy locus

(Bonduriansky and Day 2009). A study based on the design

of Waterland et al. (2007), but without continuous methyl

donor supplementation throughout generations, would be

informative in this respect. Not only dietary supplementa-

tion, but also dietary restriction can induce transgenera-

tional epigenetic effects. Burdge et al. (2007) reported

altered methylation patterns in gene promoters of adult

male F1 and F2 offspring from mother rats fed on a protein-

restricted diet during pregnancy.

Exciting work on transgenerational epigenetic effects

has been performed with plants. Lang-Mladek et al. (2010)

recently showed that temperature and UV-B stress cause

changes in the histone occupancy and histone acetylation

on H3 in a reporter gene in Arabidopsis. These alterations

were transmitted to non-stressed progeny, but limited to

areas consisting of a small number of cells and to a

restricted number of generations. Other epigenetic mech-

anisms were also shown to be affected in untreated progeny

of Arabidopsis exposed to stress: DNA methylation was

altered in non-exposed progeny of salt-, heat-, cold-, UV-

or flood-stressed Arabidopsis plants (Boyko et al. 2010).

Using mutants, these authors showed that this was depen-

dent on ncRNA processing enzymes. Also in this study,

effects did not persist in successive generations. In a study

with Norway spruce, Yakovlev et al. (2011) set up a

common garden experiment, as suggested by Bossdorf

et al. (2008), to determine the effect of temperature during

zygotic embryogenesis on bud phenology and cold accli-

mation. By studying the transcriptome in suppressive

subtracted cDNA libraries, they found that three genes

involved in siRNA pathways showed differential tran-

scription in progeny of plants with a cold versus warm

treatment during embryogenesis. These siRNAs were

suggested to be involved in the epigenetic memory phe-

nomenon resulting in different performance in progeny

with an identical genetic background. An interesting aspect

of this study is that different families of spruce differed in

their epigenetic memory response, making the case for a

genetic basis of this epigenetic system.

Epigenetic changes due to exposure to chemicals

Cancer research is probably the most intensely studied field

in epigenetics. The pattern of DNA methylation changes

substantially when cells become cancerous: the tumor

genome becomes globally hypomethylated, while local and

discrete regions at the promoter region of tumor-suppressor

genes undergo intense hypermethylation (Ropero and

Esteller 2009). Exposure to non-genotoxic carcinogens has

been shown to induce DNA methylation changes. One

well-known example is phenobarbital, which was shown to

reduce the overall methylation level in the liver of a tumor-

sensitive mouse strain, while increasing DNA methylation

in GC-rich regions of hepatic DNA (Counts et al. 1996;

Watson and Goodman 2002). Wu et al. (2010) recently

reported that maternal exposure to the non-genotoxic car-

cinogen DEHP (Di-2-(ethylhexyl) phthalate), which is

widely distributed in the environment due to its use as

plasticizer, caused increased DNA methylation in testes of

fetal and newborn mice. This was associated with the

induction of testicular dysgenesis syndrome. Newbold et al.

(2006) described an increased incidence of tumors in mice

that were prenatally or neonatally exposed to the synthetic

estrogen diethylstilbestrol (DES). This was also observed

in the offspring of early DES exposed female mice mated

with unexposed sires. Transgenerational transfer of altered

DNA methylation was suggested to be involved in the

increased tumor rate in non-exposed offspring, since DES


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exposure was shown to induce altered methylation in

several uterine genes (Li et al. 2003; Bromer et al. 2009).

Based on such studies with rodents or mammalian cells,

Aniagu et al. (2008) investigated the effects of exposure to

the putative non-genotoxic carcinogens 17b-oestradiol (E2)

and hexabromocyclododecane (HBCD) and the demethy-

lating agent 5-aza-20-deoxycytidine (5-AdC) on global

DNA methylation levels in liver and gonads of the three-

spine stickleback Gasterosteus aculeatus. This is one of the

few studies that have addressed the epigenetic impact of

environmental exposure to chemicals in species different

from humans, primates or rodents. An overview of these

studies, which will be further discussed in this section, can

be found in Table 1. HPLC was used to measure 5-methyl-

20-deoxycytidine levels in the stickleback DNA. HBCD

caused no significant effects. E2 exposure caused a non-

significant decrease in DNA methylation in female livers,

while a significant increase in DNA methylation in gonads

of exposed males was observed. Unexpectedly, 5-AdC,

which is a known inhibitor of DNA methyltransferases and

which was used in this study as a positive control for

demethylation, also caused an increase in DNA methyla-

tion in male gonads, while the expected decrease in DNA

methylation was observed in female liver samples. The

authors speculate that a rebound hypermethylation fol-

lowing cytotoxicity or a secondary response to factors

released from other tissues in which hypomethylation may

have taken place could be the cause of this unexpected

hypermethylation. A large knowledge gap remains to be

filled regarding the (transgenerational) epigenetic effects of

non-genotoxic carcinogens in ecotoxicologically relevant

species.

Air pollution can induce alterations in global DNA

levels. In sperm of mice exposed to polluted air near steel

plants or a highway, an increase of global methylation was

reported by Yauk et al. (2008). These authors also observed

DNA strand breaks and they suggest that this caused

increased DNMT activity—DNMTs are known to be

upregulated during DNA damage and bind with high

affinity to many DNA lesions—resulting in the observed

hypermethylation. Tarantini et al. (2009) described reduced

DNA methylation levels in Alu and LINE-1 repeats

(monitored as an indicator for global DNA methylation) in

leukocytes from workers in a steel plant who were exposed

to well defined levels of PM10 (Particulate Matter with

aerodynamic diameters \ 10 lm). These results are in

accordance with the observation of decreased LINE-1

DNA methylation in human blood samples after airborne

exposure to black carbon or PM2.5 (Baccarelli et al. 2009).

An interesting unifying hypothesis for explaining hy-

pomethylation due to either nutritional factors or environ-

mental chemical exposure was recently proposed by Lee

et al. (2009). These authors suggested that the impairment

of S-adenosyl-methionine (SAM, Fig. 1a) synthesis may be

at the basis of these methylation reducing pathways. A diet

low in methyl donors inhibits the methyl addition to

homocysteine to form methionine, which is one of the

building blocks of SAM. However, homocysteine can also

be used to form glutathione (GSH). Glutathione is conju-

gated with toxic chemicals or their metabolites in the phase

II detoxification pathway. Exposure to toxicants may

increase the need for GSH, exploiting homocysteine for

GSH synthesis and not for SAM synthesis, resulting in

lower SAM levels and hypomethylation. Wang et al.

(2009) reported a dose-dependent decrease in global DNA

methylation in the liver of the false kelpfish Sebastiscus

marmoratus after a 48-day exposure to waterborne tribu-

tyltin, triphenyltin or a mixture of both. This was induced

by altering SAM and SAH (S-adenosyl homocysteine)

concentrations, and especially the SAM:SAH ratio, rather

than affecting DNMT1 expression. TBT is indeed known

to decrease cellular GSH concentrations (Huang et al.

2005). Impaired SAM synthesis may also be the reason for

a decreasing trend of global DNA methylation at Alu

repeats with increasing concentrations of persistent organic

pollutants (POPs), which was observed in a study of blood

samples from Greenlandic Inuit (Rusiecki et al. 2008).

One of the earliest papers discussing the effects of

exposure to chemicals on an epigenetic system in an

environmentally relevant organism also dealt with organic

pollutants (Shugart 1990). This study reported persistent

hypomethylation in the DNA of bluegill sunfish upon

exposure to benzo[a]pyrene, metabolites of which are

known to be conjugated with GSH (Jernstrom et al. 1996).

However, other mechanisms were suggested to cause the

hypomethylation: the onset of DNA hypomethylation cor-

related with DNA strand breakage, which suggested an

inefficient methyltransferase system. In a later phase,

hypomethylation corresponded to the appearance of

benzo[a]pyrene-DNA adducts, which is known to inhibit

the maintenance of DNA methylation.

Environmental exposure to several metals is known to

induce epigenetic changes. Nickel (Ni) is a known car-

cinogenic, but its genotoxicity is low. Lee et al. (1998)

demonstrated that Ni induces overall genomic DNA hy-

permethylation in a transgenic hamster cell line, although

the DNA methyltransferase enzyme activity was inhibited

by Ni exposure in vitro and in vivo. This lead to the model

that Ni2? directly interacts with heterochromatin, replacing

Mg2? in the phosphate backbone, which results in

increased chromatin condensation. The enhanced chroma-

tin condensation capacity of the Ni2? ions would then

attract and condensate neighboring DNA that was previ-

ously in a euchromatin state. This newly formed hetero-

chromatin can subsequently be targeted by de novo DNA

methyltransferases. In cell lines, crystalline forms of Ni are


123

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123

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123

more readily delivered to the nucleus than soluble Ni.

Exposure to crystalline Ni forms, e.g. by inhaling small

particles, would thus form a greater hazard than exposure

to soluble Ni forms, which would be dissipated by disso-

lution (Costa et al. 2005). Opposed to this heterochromatin

inducing effect of Ni, a study by Zhou et al. (2009) dem-

onstrated an increase in H3K4 tri-methylation, an epige-

netic mark associated with euchromatin, in human

carcinoma cells upon exposure to Ni.

Cadmium (Cd) exposure for 1 week caused hypome-

thylation in a rat liver cell line, while a 10 week exposure

period induced DNA hypermethylation (Takiguchi et al.

2003). Cd was shown to inhibit DNA methyltransferase

activity, possibly caused by binding with the DNA binding

domain of the enzyme, which can explain the initial

hypomethylation. The hypermethylation after longer Cd

exposure co-occurred with an increased DNA methyl-

transferase activity. This was suggested to be a compen-

satory effect: because of the initial inhibition of the enzyme

activity, DNA methyltransferase genes are overexpressed,

leading to the observed hypermethylation. This is consis-

tent with other studies, in which short-term (24–48 h) and

long-term (2 months) Cd exposure induced hypomethyla-

tion and hypermethylation, respectively, in human cells

(Huang et al. 2008; Jiang et al. 2008).

Another metal for which exposure has been associated

with epigenetic effects is arsenic (As). As is methylated

during its metabolism, with S-adenosyl-methionine acting

as a methyl donor. Thus, As metabolism interferes with the

one-carbon metabolism pathway, which is essential for

DNA methylation. In a study with rat liver cell lines

chronically exposed to As, global hypomethylation was

indeed observed and shown to be dose- and time-depen-

dent. Depressed levels of S-adenosyl-methionine were

observed concurrently (Zhao et al. 1997). In a recent study

with immortalized human urothelial cells exposed to As,

results from DNA methylation microarrays demonstrated

non-random alterations in DNA methylation at multiple

promoters and CpG rich sites in the genome (Jensen et al.

2009). These aberrations were both hypo- and hyperme-

thylation events. The authors demonstrated that As did not

affect DNMT activity, but the mechanism by which these

site-specific changes occur remains to be found. Similar to

Ni, arsenate exposure was also shown to induce histone

trimethylation at H3K4 in human carcinoma cells (Zhou

et al. 2009). Finally, Marsit et al. (2006) showed that

arsenic exposure significantly altered miRNA expression

profiles in exposed human lymphoblastoid cells, resulting

in a global increase of miRNA expression.

Lead (Pb) is a metal which has only recently been studied

in an epigenetic context. Wu et al. (2008) reported a

decreased DNA methyltransferase activity (1) in mouse

cortical neuronal cells exposed for 24 h to Pb and

subsequently aged 1 week and (2) in brain cells from

23-year old monkeys that were exposed to Pb during the

first 400 days of their lives. In accordance with this inhib-

itory effect of Pb, Pilsner et al. (2009) demonstrated an

inverse relationship between Pb concentrations in maternal

bone tissue, which are good bio-indicators of Pb exposure,

and global DNA methylation in umbilical cord blood

samples in a study group of Mexican people. The mecha-

nism of Pb interaction with DNA methylation is unknown.

Following a similar approach, but in an ecotoxicological

context, the same group of authors used a luminometric

methylation assay (LUMA) to study global DNA methyl-

ation in brains of polar bears that were hunted in Greenland

during 3 years (Pilsner et al. 2010). In male bears, they

observed an inverse relationship between DNA methyla-

tion and brain mercury (Hg) exposure levels, of which the

significance level was p = 0.07. There was a large varia-

tion in DNA methylation levels in the studied population of

47 bears, also between animals with comparable Hg con-

centrations in the brain. In this type of field studies, other

unknown factors may also influence DNA methylation.

The observed trend suggested a possible Hg-induced hy-

pomethylation in male bears. However, further studies are

required to confirm this finding.

A unifying process to account for the effect of different

metals on DNA methylation may be metal-induced oxi-

dative stress. Metals are known to catalyze and increase the

production of reactive oxygen species, which may create

oxidative DNA damage. This can inhibit the action of DNA

methyltransferases and as such lead to hypomethylation

(Baccarelli and Bollati 2009). This process was initially

thought to be a potential cause of the metal concentration-

dependent global decrease in DNA methylation observed

in a study with white clover Trifolium repens and industrial

hemp Cannabis sativa exposed to mixtures of nickel,

chromium and cadmium (Aina et al. 2004). Interestingly,

however, the hypomethylation in these plants was not

random but involved specific DNA sequences, which

reproducibly lost methylation in all replicates per treat-

ment. Moreover, hypermethylation events were also

detected at specific loci. These results showed that epige-

netic responses to metal stress are more diverse than what

can be explained by metal-induced reactive oxygen spe-

cies. This also follows from the increased overall DNA

methylation observed in the liver of the goldfish Carassius

auratus when exposed to copper, zinc, lead, cadmium or a

metal mixture; with lead and cadmium inducing a larger

change than copper and zinc (Zhou et al. 2001). DNA

methylation changes may be part of the defense mechanism

against metal stress. Identifying the genes affected by these

epigenetic modifications and linking them to alterations at

the transcriptional or proteomic level is needed to elucidate

the nature of metal-induced changes in DNA methylation.


123

A key study demonstrating the transgenerational effects

of toxicant exposure was performed by Anway et al.

(2005). These authors exposed pregnant rats (F0 genera-

tion) to the fungicide vinclozolin or the pesticide

methoxychlor, which are both endocrine disrupting chem-

icals. Male F1 offspring of these rats exhibited a reduced

spermatogenic capacity (lower sperm counts and mobility).

These effects were observed in three subsequent genera-

tions of non-exposed male offspring (F2 to F4) and were

associated with aberrant DNA methylation patterns in

sperm. Transgenerational transfer of DNA methylation was

suggested to be the mechanism of transmission of these

effects, through altering the transcriptome (Anway et al.

2008). Next to the effect on fertility, vinclozolin exposure

of gestating F0 females also induced an increased tumor

incidence rate and increased frequencies of different dis-

ease states (e.g. of the kidney and immune system) in aging

F1 to F4 males (Anway et al. 2006). In later studies it was

shown that F1 to F4 male offspring also suffered from

several abnormalities in the ventral prostate. The prostate

transcriptome of affected F3 males showed differential

expression compared to control males for 259 genes, which

was suggested to be the consequence of the altered DNA

methylation pattern (Anway and Skinner 2008). Female F1

to F3 progeny of vinclozolin exposed pregnant rats also

exhibited increased adverse effects compared to control

rats, including kidney abnormalities, tumor development

and uterine hemorrhage and/or anemia during late preg-

nancy. While outcross experiments demonstrated that

effects in males were passed on through the male germline,

effects in females were only observed when both parents

were descendants of vinclozolin-exposed rats (Nilsson

et al. 2008). Epigenetic mechanisms were not studied in

these female rats, but were suggested to have caused the

transgenerational disease states.

The vinclozolin studies evoked some controversy, which

was fed by the recent retraction by the same research group

of a paper describing DNA sequences with altered DNA

methylation in the sperm of offspring of vinclozolin-

exposed rats. These sequence results, which were generated

solely by the first author, could not be reproduced (Chang

et al. 2006, 2009). However, the original results from

Anway et al. (2005) were not involved in this retraction. In

this work, 25 DNA fragments were reported to be subject

to altered methylation upon in utero exposure to vincloz-

olin of F1 rats. Two of these fragments were more thor-

oughly investigated and also showed altered DNA

methylation patterns in F2 and F3 rats. In a follow-up test

with oral administration of vinclozolin to pregnant rats,

which was deemed more relevant in terms of risk assess-

ment, no effects were observed in F1 to F3 offspring

(Schneider et al. 2008). Lower toxicant concentrations at

the site of action compared to the concentrations after

intraperitoneal injection in the study of Anway et al. (2005)

may play a role in the different outcomes of both studies. In

another verification study with the same intraperitoneal

injection of vinclozolin to pregnant rats as in Anway et al.

(2005), no effects on spermatogenesis in F1 rats and no

alterations in DNA methylation of F1 and F2 rats were

measured (Inawaka et al. 2009). This different finding may

be due to a different genetic background of the rats. In a

repeat experiment, the outbred status of the rat line, as well

as the exposure timing and duration were found to be

crucial to the effects (Skinner et al. 2010). Interestingly, in

a recent study with mice, which were administered a dose

of 50 mg/kg/day vinclozolin during pregnancy, transgen-

erational effects on the DNA methylation status of some

imprinted genes in sperm were observed in the F1, F2 and

F3 generations (Stouder and Paoloni-Giacobino 2010).

Although the intensity of the effect gradually reduced over

the generations, this study confirms the induction of

transgenerational effects on DNA methylation due to in

utero vinclozolin exposure.

In an (eco)toxicological context, the concentration-

effect relation should not be neglected. Anway et al. (2006)

reported that a dose of 50 mg vinclozolin/kg/day produced

‘more variable’ effects than the dose of 100 mg/kg/day

used in their transgenerational study. Other factors, related

to the exposure to the substance, e.g. the half live and

application rate/time of a pesticide, are also important

when this type of effects are translated to field situations. In

an interesting exercise, Dalkvist et al. (2009) modeled the

long-term impact of a vinclozolin-like pesticide on vole

populations in a landscape setting, taking into account the

epigenetic transgenerational effect. Results demonstrated

that animal ecology and behavior (e.g. habitat preference)

were equally important as toxicology (e.g. No Observed

Effect Level) to determine population-level impacts.

A series of recent publications reported on epigenetic

and transgenerational effects of toxic stress in the water

flea Daphnia magna, an important ecotoxicological model

species. DNA methylation was detected at CpG sites in two

unknown genomic fragments. Exposure to an elevated Cd

concentration for two generations, which resulted in

decreased reproduction, did not affect the DNA methyla-

tion at these sites (Vandegehuchte et al. 2009a). Subse-

quently, these authors quantified DNA methylation in

D. magna exposed to zinc in a three-generation study. In

the F1 generation offspring of F0 generation Zn exposed

daphnids, a slight but significant decrease in DNA meth-

ylation was observed. This demonstrated that environ-

mental conditions can affect DNA methylation in this

species. However, this effect did not propagate into the

next generation (Vandegehuchte et al. 2009b). The effect

of this Zn exposure on gene transcription was also studied

using custom cDNA microarrays. The transcription of a


123

large number of genes was affected by the Zn exposure,

both in the exposed daphnids and in their non-exposed

offspring. A large proportion of common genes were

similarly up- or downregulated in the exposed and non-

exposed F1 daphnids, suggesting a possible effect of the

DNA hypomethylation. Interestingly, two of these genes

can be mechanistically involved in DNA methylation

reduction. The similar transcriptional regulation of two and

three genes in the F0 and F1 exposed daphnids on one hand

and their non-exposed offspring on the other hand, could be

the result of a one-generation temporary transgenerational

epigenetic effect. A stable transgenerational epigenetic

effect on gene transcription through the three generations,

however, was not observed (Vandegehuchte et al. 2010c;

Vandegehuchte et al. 2010a). It should be stressed though,

that gene-specific links between DNA methylation and

transcription could not be made in these studies, since only

global methylation was measured. With the arrival of a

D. magna genome assembly, which is work in progress by

the Daphnia Genomics Consortium (http://daphnia.cgb.

indiana.edu), locus-specific DNA methylation analyses

will enable deciphering the transcriptional consequences

and potential phenotypic implications of altered DNA

methylation.

Finally, the effect of substances known to affect DNA

methylation in mammals was evaluated using D. magna

multigeneration experiments. Vinclozolin and 5-azacyti-

dine, a demethylating drug, were shown to induce DNA

hypomethylation in the first exposed generation (F0).

Opposed to the results of the above described studies with

rats, the vinclozolin-induced effect did not propagate into

the next generation. In the case of 5-azacytidine exposure,

however, this effect was transferred to the two subsequent

non-exposed generations, coinciding with a significantly

reduced juvenile growth (Vandegehuchte et al. 2010b).

These results demonstrate the possibility of transgenera-

tional inheritance of environment-induced epigenetic

effects in non-exposed subsequent generations of

D. magna, which is an important principle. Key issues for

the ecotoxicological implications of this, which need fur-

ther study, are the causality between DNA methylation and

the reduced growth phenotype, the ecological relevance of

this reduced growth and the potential more subtle site-

specific effects on DNA methylation at exposure concen-

trations lower than 2.9 mg/l.

Another recent report on multigenerational epigenetic

effects described DNA methylation changes in the dande-

lion Taraxacum officinale exposed to different kinds of

environmental stress (Verhoeven et al. 2010). Similar to

parthenogenetic D. magna, apomictic T. officinale offer the

advantage of detecting epigenetic variation in the absence

of genetic variation. Nutrient limitation, salt stress and the

application of plant hormones each resulted in an increased

epigenetic variation in terms of locus-specific DNA

methylation. Many of these modifications in DNA meth-

ylation were transmitted to the offspring which was raised

in an unstressed environment, and which also exhibited

significantly affected traits. It would be most interesting to

investigate the transmission of these effects into sub-

sequent generations and to test the effects of environmental

exposure to chemicals other than plant hormones in this

species.

These type of studies incite to investigate the epigenetic

effects of substances with known transgenerational effects,

such as endocrine disrupting chemicals. Brown et al.

(2009) reported reduced embryonic survival in the rainbow

trout Oncorhynchus mykiss due to exposure of males to the

endocrine disrupting chemical 17a-ethynilestradiol (EE2)

during late spermatogenesis. These authors discussed that

this was potentially mediated by an epigenetic mode of

action. However, they did not evaluate the molecular

mechanism of EE2 toxicity.

Adult zebrafish (Danio rerio) were exposed to EE2 by

Stromqvist et al. (2010) in order to study the effect on DNA

methylation in specific CpG sites in the 50 flanking region

of the vitellogenin I gene in liver and brain. This specific

gene was selected because of the known induction of

vitellogenin by environmental estrogen exposure in zeb-

rafish. Pyrosequencing revealed that the DNA methylation

levels in the liver at all three investigated sites for females

and at two sites for males were significantly reduced upon

EE2 exposure, suggesting that demethylation may be

involved in vitellogenin induction. The authors recommend

future ecotoxicological studies on epigenetic changes in

fish. It would be very interesting to determine the DNA

methylation in an expanded zebrafish experiment with a

next exposed and non-exposed generation and to include

the analysis of phenotypic effects. EE2 is known to induce

developmental effects at lower concentrations in offspring

from EE2-exposed zebrafish, compared to concentrations

causing effects in the parental generation (Soares et al.

2009). If multigenerational DNA methylation changes are

involved, this may also impact non-exposed offspring.

The soy isoflavone genistein, also a known endocrine

disruptor, has been shown to reduce DNA methyltransfer-

ase activity, but the detailed mechanism of how it affects

DNA methylation has yet to be determined (Majid et al.

2010; Ross 2009). Day et al. (2002) demonstrated that male

mice fed a genistein-enriched diet showed increased DNA

methylation at specific genes in the prostate. Dietary gen-

istein supplementation in female mice also lead to

increased methylation at the Avy metastable epiallele in

offspring, which was associated with a shift in coat color,

similar to what was observed with methyl donor supple-

mented food (Dolinoy et al. 2006). In a study with the

ecotoxicological model species Daphnia magna exposed to


123

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http://daphnia.cgb.indiana.edu

chemicals with a known effect on DNA methylation,

however, no global DNA methylation changes were

observed upon genistein exposure via water (Van-

degehuchte et al. 2010b).

Nanomaterials and epigenetic changes

In the last decades, the production and use of nanomaterials

has increased dramatically. The special properties that

these materials possess because of their unique physical

and chemical structure have also raised questions related to

their possible impact on human health and the environ-

ment. In recent years, epigenetics has been introduced in

the field of nanotoxicology. Choi et al. (2008) demon-

strated global histone hypoacetylation in human breast

carcinoma cells exposed to cadmium telluride quantum dot

nanoparticles at very low concentrations. This was linked

to decreased gene transcription. Global hypoacetylation

was suggested to induce a global reduction in gene tran-

scription, including anti-apoptotic genes. That could be an

explanation for the observed cell death after quantum dot

exposure. However, some anti-apoptotic genes were shown

to be upregulated in the presence of the quantum dots. The

authors suggest further studies to determine the global

reconfiguration of chromatin in the exposed cells.

Exposure to silicon-dioxide (SiO2) nanoparticles at

concentrations up to 10 lg/ml induced a dose-dependent

hypomethylation in the DNA of human HaCaT cells (Gong

et al. 2010). Interestingly, SiO2 microparticles at the same

concentration of 10 lg/ml did not induce a significant

effect on DNA methylation. This observation coincided

with a dose-dependent decrease in the expression at mRNA

and at protein level of the DNA methyltransferases

DNMT1 and DNMT3a and the methyl-binding domain

protein MBD2. The authors refer to their study as ‘an an

initial step in the unexplored field of epigenetic changes by

nanoparticles’. Knowledge from this field should be taken

into account in the developing research discipline of

nanoecotoxicology, where no epigenetic research has been

performed yet (Baun et al. 2008).

Epigenetics and risk assessment of chemicals

The above-described vinclozolin studies highlight potential

consequences of environmental chemical exposures for the

health of non-exposed future generations. This may be

relevant for human health and toxicology, which has been

described in popular media in expressions like ‘what you

eat or drink today could affect the health of your great-

grandchildren (Watters 2006)’. However, this concept

could also be of importance for ecotoxicology and envi-

ronmental risk assessment of chemicals, as indicated by

the transgenerational effect of 5-azacytidine in Daphnia.

Indeed, if chemical exposure to one generation can have

effects on multiple subsequent non-exposed generations,

the risk assessment of these chemicals should incorporate

this time interval between effects and exposure in previous

generations (Fig. 2).

The fact that exposure to chemicals can induce epige-

netic changes, combined with the occurrence of transgen-

erational epigenetic effects has raised concern about the

role of epigenetics in chemical safety/risk assessment. In

2009, the Health and Environmental Sciences Institute of

the International Life Sciences Institute organized a

workshop in Research Triangle Park (North Carolina) to

discuss the state of the science regarding epigenetic chan-

ges, with the aim of assessing the knowledge needed to

incorporate an epigenetic evaluation into safety assess-

ments (Goodman et al. 2010). The workshop report con-

cludes that a great deal needs to be learned prior to taking

the science-based decision to incorporate epigenetics into

safety assessment. This also holds true for the environ-

mental risk assessment of chemicals. Several recommen-

dations for future studies were made during that workshop.

The choice of an appropriate exposure route and relevant

exposure doses/concentrations is a very important aspect

when evaluating epigenetic effects of chemical exposure. It

is also key that any changes detected in an epigenetic

system are rationally linked to adverse phenotypical

effects. In the framework of chemical safety or risk

assessment, ideally there should be information about (1)

Fig. 2 Conceptual figure indicating that exposure to chemicals can

induce epigenetic changes, e.g. demethylation of DNA (represented

by the removal of black dots from the schematic DNA double helix)

by inhibiting the action of DNA methyltransferases (DNMT) and

phenotypic effects (schematically represented by the black fins).

These epigenetic changes and/or phenotypic effects may or may not

be transmitted to the next generation, which is not exposed to the

chemical. If these changes persist for multiple generations (shadedframe), non-exposed organisms would experience effects of their

ancestors’ exposure, which would have profound implications for

environmental risk assessment of chemicals


123

molecular mechanisms, (2) the epigenetic changes result-

ing from this mechanism and (3) the integrated phenotypic

effects of these changes. Basic research, including the

determination of reference epigenomes, experiments with

positive and negative controls and the setting of threshold

and cutoff values are required to fill data gaps with respect

to these information needs. Similar conclusions and rec-

ommendations were made in a review on epigenetics and

chemical safety assessment focused on human health risks

by LeBaron et al. (2010). These authors also argued that

the current toxicological testing battery is expected to

identify any potential adverse effects, including those

resulting from epigenetic changes. The question of species

cross-relevance, which is also highly relevant to ecotoxi-

cological studies, was emphasized in this review. Studies

comparing the methylation status of imprinted genes

(considered to be the most conserved) in mice versus man

showed that there is a high level of discordance and that

species are highly dissimilar. Hence, epigenetic effects

observed in one species cannot be assumed to occur in all

other species.

Up to now, evolutionary aspects or effects of chemical

exposure in future non-exposed generations are not con-

sidered in the ecological risk assessment process. It is not

known how much uncertainty this implies for the current

environmental risk assessments. However, the limited

number of reports reviewed above (Table 1) are a cause for

concern. Before recommendations can be made to incor-

porate epigenetics into the ecological risk assessment

process, more fundamental research is needed to address

(1) the occurrence of chemical-induced epigenetic modi-

fications at environmentally realistic exposure concentra-

tions in ecotoxicologically relevant species, (2) the

phenotypic and population-level effects of these modifi-

cations and (3) the transmission of these changes to sub-

sequent non-exposed generations.

Concluding remarks

It is now clear that environmental exposure to chemicals

can affect the epigenetic status of several species, not only

rodent models, but also ecotoxicologically relevant

fish, plants and invertebrates (Table 1). Moreover, certain

studies demonstrated epigenetic alterations in the non-

exposed offspring of exposed organisms. This is a new

concept in ecotoxicology. If chemical exposure to one

generation can have effects on multiple subsequent

non-exposed generations, the risk assessment of these

chemicals should incorporate this time interval between

effects and exposure in previous generations. However,

major knowledge gaps in most of the reported ecotoxico-

logical studies are (1) the potential causal links between

chemical-induced epigenetic changes and phenotypic

effects and (2) the persistence of these changes and their

higher-level effects into multiple subsequent generations.

In that respect, the evaluation of classic ecotoxicological

endpoints will remain necessary, but should be comple-

mented by molecular research. The wider availability of

relatively new technologies such as next-generation

sequencing can help to understand the functional conse-

quences of epigenetic modifications, e.g. through a gen-

ome-wide investigation of affected genes in case of global

demethylation, and to design focused experiments to

determine higher-level impacts of epigenetic changes.

The advent of epigenetics in all areas of biological

sciences means that ecotoxicologists should consider

altering experimental protocols to include developmental

exposure, epigenetic analysis and transgenerational studies,

as suggested by Legler (2010). With this, basic ecotoxi-

cological concepts such as bioavailability, exposure routes

and relevant concentration/effect relations should be taken

into account. With the current knowledge it is too early to

assess the full impact of epigenetics on ecotoxicology or

ecological risk assessment of chemicals. However, the state

of the science in this field evolves at a rapid pace and

results presented in this review are anticipated to encourage

the scientific community to further the understanding of

epigenetics in an ecotoxicological context.

Acknowledgments The authors wish to thank two anonymous

reviewers and the editor dr. Richard Handy for their constructive

comments which improved the manuscript.

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